1. Technical Field of the Disclosure
The present disclosure is directed, generally, to the detection of fungal pathogens in a patient sample. More specifically, provided herein are quantitative PCR-based compositions and methods for the diagnosis of invasive pulmonary aspergillosis (IPA) in a patient sample, such as bronchoalveolar lavage (SAL) fluid. The methods presented herein involve isolating a sample, collecting a cell fraction from the sample, extracting DNA from the cell fraction, carrying out a quantitative PCR (qPCR) reaction on the sample to generate an amplicon that includes a region of an Aspergillus spp. gene (such as a ribosomal RNA (rRNA) gene), and detecting the PCR amplicon. The present disclosure also provides primers, primer sets, and kits for specifically detecting an Aspergillus spp. fungal pathogen in the presence of human DNA, including ribosomal DNA (rDNA).
2. Description of the Related Art
Invasive pulmonary aspergillosis (IPA) is a common infection in patients with hematological malignancies and those undergoing hematopoietic cell transplantation [1]. Despite the availability of new mould-active antifungal medications such as extended spectrum azoles (e.g., voriconazole and posaconazole) and echinocandins, aspergillosis remains a significant cause of death in patients with cancer [2]. Delays in the institution of appropriate antifungal therapy may contribute to the high mortality seen with IPA, and the diagnosis of aspergillosis remains a clinical challenge, enhancing the potential for delay [1, 3, 4].
Most symptoms of IPA are non-specific, such as fever, cough, or chest pain, and many patients have no symptoms at all. Although some radiographic findings in the lungs can suggest aspergillosis, such as the presence of a halo sign (ground glass opacity surrounding a nodule) or cavitating nodules, these findings can also be found in subjects with pulmonary zygomycosis or other infections and, thus, are not necessarily specific [5]. The failure to make an accurate diagnosis frequently results in the use of empirical antifungal therapy in the suitable immunocompromised host.
The diagnosis of IPA remains challenging. Bronchoalveolar lavage (BAL) fluid is routinely used to assess the presence of fungi at the site of pulmonary infection. Conventional microbiological techniques like culture and histology of BAL fluid are most commonly used for the diagnosis of IPA, but have suboptimal sensitivity and, in the case of culture, may take several days [6-8]. Detection of the fungal cell wall constituents like galactomannan (in serum and BAL fluid) and beta-glucan (in serum) are promising diagnostic alternatives to facilitate the diagnosis of invasive fungal infection, but false positive and false negative results remain problematic with both assays [9-11].
Molecular diagnostic techniques such as nucleic acid detection by PCR are emerging as potentially more sensitive and rapid alternatives to conventional techniques for the diagnosis of IPA [12-19], but published studies lack key quality control standards that are useful in identifying problems with false negative and false positive results within a study. Furthermore, the lack of appropriate controls affects the ability to coherently compare different published diagnostic PCR platforms for IPA [12, 21, 22, 25].
Quantitative PCR has several advantages when used for the detection of Aspergillus spp. First, qPCR is highly sensitive with the potential to detect a few gene copies per reaction, or less than a single genome for multicopy genes such as the rRNA gene. Second, by taking advantage of both conserved and variable regions of genes, primers and probes can be made that are specific for a given genus, species or strain of microbe. Third, qPCR can measure the amount of microbial DNA in a clinical sample, which may be useful for assessing the burden of infection and in distinguishing between colonization and infection. Fourth, multiplexed qPCR reactions can reduce the necessity of running independent qPCRs allowing for the detection of multiple targets or for inclusion of amplification controls in a single reaction. Fifth, qPCR assays can be completed in a few hours, resulting in a rapid turn around time for reporting results.
To develop an optimal qPCR assay for diagnosis, however, several challenges and shortcomings must be addressed to minimize false positive and false negative results [20-22]. False negatives can occur due to suboptimal DNA extraction (i.e. low recovery of DNA and/or the presence of PCR inhibitors), large quantities of human genomic DNA competing with the microbial target for amplification, and suboptimal analytical sensitivity of the qPCR reaction itself (high detection threshold). False positives can occur due to introduction of contamination during sample collection, DNA extraction, and PCR set-up, resulting from the presence of fungi in the environment or fungal PCR product carry-over. In addition, false positives can occur in the setting of suboptimal analytical specificity in the qPCR, resulting from cross-reactivity of the target qPCR assay with other (non-target) fungi or DNA. Accordingly, optimal qPCR assays for IPA should incorporate controls to assess for the factors contributing to false positive and false negative results.
What is critically needed in the art are compositions and methods for achieving the diagnosis of invasive pulmonary aspergillosis. Ideally, such compositions and methods would employ quality control measures to address false positives and negatives which can hinder accurate evaluation of diagnostic performance.